JP6360848B2 - Rapid mass screening test for lysosomal disease 3 responsible enzyme - Google Patents
Rapid mass screening test for lysosomal disease 3 responsible enzyme Download PDFInfo
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Landscapes
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
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Description
本発明は、ライソゾーム病３疾患（ファブリー病、ポンペ病、ゴーシェ病）の責任酵素の迅速マススクリーニング検査法に関する。 The present invention relates to a rapid mass screening test for responsible enzymes for three lysosomal diseases (Fabry disease, Pompe disease, Gaucher disease).
細胞内小器官の一つであるライソゾーム（lysosome；「リソゾーム」、「リソソーム」とも呼ばれる。）は、細胞の中で糖質や糖脂質の分解を行っている。これには約60種類の加水分解酵素が関与しており、それらの責任酵素の欠損・異常によって、ライソゾームの分解機能が発揮されなくなると、本来分解されるべき物質が老廃物として体内に蓄積してしまう。このようにして生じる先天性代謝異常症の総称がライソゾーム病である。欠損している酵素によって症状が異なり、ファブリー病、ポンペ病、ゴーシェ病など、約30種類の病名で知られる。 One of the organelles, lysosome (also called “lysosome” or “lysosome”) degrades carbohydrates and glycolipids in cells. This involves about 60 types of hydrolases, and if the lysosomal degradation function fails to function due to the deficiency / abnormality of these responsible enzymes, substances that should be degraded accumulate in the body as waste products. End up. The general term for inborn errors of metabolism that arise in this way is lysosomal disease. Symptoms vary depending on the deficient enzyme and are known by about 30 types of disease names such as Fabry disease, Pompe disease, and Gaucher disease.
ライソゾーム病は、別の先天性代謝異常症であるフェニルケトン尿症のように、摂取制限で治療できるような必須アミノ酸代謝異常とは異なり、それらの責任酵素は生体内で合成されることから、根治には遺伝子治療が必要である。現在は、酵素補充療法のための薬剤が市販され、多くの場合、酵素補充療法の早期治療開始により、患者のQOL向上や発症の予防など臨床的改善がもたらされる。このため、これらの疾患が新生児マススクリーニング検査によって早期に発見されることが望まれている。 Unlike essential amino acid metabolism disorders that can be treated with restricted intake, such as phenylketonuria, which is another inborn error of metabolism, lysosomal disease is because their responsible enzymes are synthesized in vivo, Complete therapy requires gene therapy. Currently, drugs for enzyme replacement therapy are on the market, and in many cases, early initiation of enzyme replacement therapy results in clinical improvements such as improved patient quality of life and prevention of onset. For this reason, it is desired that these diseases be detected early by a newborn mass screening test.
新生児マススクリーニング検査では、血液を染み込ませて乾燥させたろ紙（以下、「乾燥血液ろ紙」という。）を用い、合成基質法による酵素活性を測定する方法が用いられる。他には、タンデム分析装置を用いた酵素活性測定法、専用装置を必要とするものの迅速に測定できるマイクロ流路測定キットなどを用いたパイロットスタディなどが実施されており、新生児マススクリーニング検査の有用性が示されている。 In the newborn mass screening test, a method of measuring enzyme activity by a synthetic substrate method using filter paper soaked with blood and dried (hereinafter referred to as “dry blood filter paper”) is used. In addition, enzyme activity measurement methods using tandem analyzers, pilot studies using microchannel measurement kits that can be measured quickly, although special devices are required, are useful for newborn mass screening tests Sex is shown.
特許文献１には、乾燥体液及び細胞組織試料中に存在するライソゾーム酵素の活性をアッセイするための方法及びキットが記載されている。特許文献１に記載の方法及びキットは、各種ライソゾーム蓄積障害を各疾患ごとに個別にアッセイするものである。 Patent Document 1 describes a method and kit for assaying the activity of lysosomal enzymes present in dry body fluids and cell tissue samples. The method and kit described in Patent Document 1 individually assay various lysosomal storage disorders for each disease.
非特許文献１には、乾燥血液ろ紙を用いたファブリー病責任酵素であるα-D-galactosidase A (GAL)の活性測定が、新生児のファブリー病スクリーニングに有効であったことが記載されている。非特許文献１ではGAL酵素のみが単独で測定の対象となっている。 Non-Patent Document 1 describes that measurement of the activity of α-D-galactosidase A (GAL), which is a responsible enzyme for Fabry disease using dry blood filter paper, was effective for Fabry disease screening in newborns. In Non-Patent Document 1, only the GAL enzyme is the object of measurement alone.
非特許文献２には、乾燥血液ろ紙及び繊維芽細胞中のポンペ病責任酵素であるacid α-glucosidase(GAA)の活性測定が、新生児のポンペ病スクリーニングに有効であること、また試料中のヘモグロビンの影響を回避する方法として水酸化バリウムと硫酸亜鉛を用いる沈殿除去法が有用であったことが記載されている。非特許文献２では、GAA酵素のみが単独で測定の対象となっている。 Non-Patent Document 2 discloses that measurement of the activity of acid α-glucosidase (GAA), a responsible enzyme for Pompe disease in dry blood filter paper and fibroblasts, is effective for screening Pompe disease in newborns, and hemoglobin in samples. It is described that the precipitation removal method using barium hydroxide and zinc sulfate was useful as a method for avoiding the influence of the above. In Non-Patent Document 2, only the GAA enzyme is the object of measurement alone.
非特許文献３には、デジタルマイクロ流路測定装置と専用キットを用いたファブリー病、ポンペ病、ゴーシェ病を含むリソソーム蓄積症（LDS）の多重スクリーニングが、新生児マススクリーニングのパイロットスタディで使用されていることが記載されている。 In Non-Patent Document 3, multiple screening of lysosomal storage diseases (LDS) including Fabry disease, Pompe disease, and Gaucher disease using a digital microchannel measurement device and a dedicated kit is used in a pilot study of neonatal mass screening. It is described that.
本発明は、新生児マススクリーニング検査におけるライソゾーム病３疾患（ファブリー病、ポンペ病、ゴーシェ病）の責任酵素活性測定を、大規模な測定機器や専用装置を必要とせず、簡便・迅速かつ安価に実施する方法を提供することを目的とする。 The present invention measures the responsible enzyme activity of three lysosomal diseases (Fabry disease, Pompe disease, Gaucher disease) in a newborn mass screening test easily, quickly and inexpensively without the need for a large-scale measuring instrument or dedicated device. It aims to provide a way to do.
新生児マススクリーニング検査は、乾燥血液ろ紙から抽出した試料を測定検体として用いる。従来のライソゾーム病責任酵素活性の測定では、検査対象となる責任酵素毎に抽出液組成が異なり、１枚の乾燥血液ろ紙からは１種類の酵素活性しか測定できず、複数の酵素を測定するには、複数枚の乾燥血液ろ紙が必要であった。また、乾燥血液ろ紙から抽出した試料の酵素活性を測定するためには、酵素反応に20時間以上が必要であり、測定期間として２日間を要していた。 In the newborn mass screening test, a sample extracted from dry blood filter paper is used as a measurement specimen. In the conventional measurement of responsible enzyme activity of lysosome disease, the composition of the extract is different for each responsible enzyme to be tested, and only one type of enzyme activity can be measured from one dry blood filter paper. Required multiple sheets of dry blood filter paper. Moreover, in order to measure the enzyme activity of the sample extracted from the dry blood filter paper, 20 hours or more was required for the enzyme reaction, and 2 days were required as the measurement period.
さらに、測定対象の責任酵素により、測定波長、操作手順、反応時間、抽出液組成、及び反応停止液組成が異なるため、多数の検体を同時に測定するためには、責任酵素毎に分析者が必要であった。 Furthermore, because the measurement wavelength, operation procedure, reaction time, extract solution composition, and reaction stop solution composition differ depending on the responsible enzyme to be measured, an analyst is required for each responsible enzyme to measure a large number of samples simultaneously. Met.
本発明者らは、上記課題を解決すべく鋭意検討を重ねた結果、ファブリー病責任酵素、ポンペ病責任酵素、及びゴーシェ病責任酵素の抽出時の酵素活性の低下や酵素活性測定時のpHに影響を及ぼさず、共通で使用できる抽出液（以下、「共通抽出液」という。）の組成を見出した。さらに、当該抽出試料が高感度に測定でき、かつ各基質液組成と共通で使用できる反応停止液（以下、「共通反応停止液」という。）の組成を見出した。 As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the enzyme activity decreases when the Fabry disease responsible enzyme, the Pompe disease responsible enzyme, and the Gaucher disease responsible enzyme are extracted and the pH when the enzyme activity is measured. The present inventors have found a composition of an extract (hereinafter, referred to as “common extract”) that can be used in common without affecting it. Furthermore, the present inventors have found a composition of a reaction stop solution (hereinafter, referred to as “common reaction stop solution”) that can be measured with high sensitivity and can be used in common with each substrate solution composition.
また、酵素活性の測定波長をシフトさせることで、乾燥血液ろ紙からの抽出試料中の蛍光物質や蛍光阻害物質の影響が少なくなることを見出し、それまで測定前に必要であった検体の遠心操作を不要とした。 In addition, we found that the effect of fluorescent substances and fluorescent inhibitors in samples extracted from dry blood filter paper was reduced by shifting the measurement wavelength of enzyme activity. Was made unnecessary.
さらに、各酵素の単位時間当たりの酵素活性は、酵素反応時間が長いほど低下する事実を見出した。さらに、酵素反応時間を従来より短時間にしても十分に測定可能な共通の条件（試料液量、基質液量、反応停止液量、酵素反応時間）を見出したことで、測定操作の作業性を向上させ、１人の分析者でも複数項目の測定を可能とした。 Furthermore, it has been found that the enzyme activity per unit time of each enzyme decreases as the enzyme reaction time increases. Furthermore, by finding common conditions (sample solution volume, substrate solution volume, reaction stop solution volume, enzyme reaction time) that can be measured sufficiently even if the enzyme reaction time is shorter than before, the workability of the measurement operation is improved. And one analyst can measure multiple items.
これらの知見に基づいて、１枚の乾燥血液ろ紙から抽出した試料より、ファブリー病責任酵素、ポンペ病責任酵素、及びゴーシェ病責任酵素の酵素活性測定を、同時、高感度、簡便、迅速、効率的かつ安価に検出する方法を見出し、本発明を完成した。 Based on these findings, the enzyme activity of Fabry disease responsible enzyme, Pompe disease responsible enzyme, and Gaucher disease responsible enzyme was measured simultaneously from a sample extracted from a sheet of dry blood filter. The present invention has been completed by finding an efficient and inexpensive detection method.
すなわち本発明は、１枚の乾燥血液ろ紙から抽出した試料を３種類の基質液を用いて酵素活性を測定することで、同時に３種のライソゾーム病を検出する方法、及び当該検査方法に使用するためのキットを提供する。ここで、本発明におけるライソゾーム病とは、ファブリー病、ポンペ病、ゴーシェ病の３疾患である。 That is, the present invention uses a sample extracted from one dry blood filter paper by measuring enzyme activity using three kinds of substrate solutions, and simultaneously using the three kinds of lysosomal diseases and the test method. Providing a kit for Here, the lysosomal disease in the present invention is three diseases of Fabry disease, Pompe disease and Gaucher disease.
したがって、本発明は以下を含む。
[１]１枚の乾燥血液ろ紙から複数のライソゾーム病責任酵素活性を迅速に測定する、ライソゾーム病責任酵素の迅速マススクリーニング検査方法であって、該責任酵素が、α-D-galactosidase A（GLA）、酸性α-glucosidase（GAA）及び酸性β-glucosidase（GBA）からなる群より選ばれる酵素であり、以下の工程を含む方法：
（１）乾燥血液ろ紙から共通抽出液を用いて血液試料を抽出する工程、
（２）抽出した血液試料に合成基質を含む基質液を添加して酵素反応を行わせる工程、
（３）酵素反応により得られた生成物を蛍光測定する工程。
[２]前記共通抽出液のpHが5.5〜6.5である、[１]に記載の方法。
[３]前記共通抽出液が、リン酸イオン及びクエン酸イオンを含む緩衝液である、[２]に記載の方法。
[４]前記共通抽出液に、0.1ｍM〜5ｍMのDithiothreitol（DTT）が含まれる、[１]から[３]のいずれか一項に記載の方法。
[５]前記共通抽出液に、マグネシウムイオン（Mg2+）が含まれる、[１]から[４]のいずれか一項に記載の方法。
[６]前記共通抽出液に、0.05％〜0.5％のTritonX-100及び0.02％〜0.1％のアジ化ナトリウムが含まれる、[１]から[５]のいずれか一項に記載の方法。
[７]前記基質液が、（i）N-アセチルガラクトサミン及び4-Methylumbelliferyl合成基質を含む緩衝液、（ii）アカルボース及び4-Methylumbelliferyl合成基質を含む緩衝液並びに（iii）4-Methylumbelliferyl合成基質を含む緩衝液の組み合わせである、[１]から[６]のいずれか一項に記載の方法。
[８]前記（i）における4-Methyumbelliferyl合成基質が、4-methylum-belliferyl-α-D-galactopyranosideであり、1ｍM〜10ｍMで含まれる、[７]に記載の方法。
[９]前記（i）における基質液のpHが4.0〜5.0である、[７]又は[８]に記載の方法。
[１０]前記（i）における基質液が、クエン酸-リン酸ナトリウム緩衝液である、[７]から[９]のいずれか一項に記載の方法。
[１１]前記（ii）における4-Methyumbelliferyl合成基質が、4-methylum-belliferyl-α-D-glucopyranosideであり、1ｍM〜10ｍMで含まれ、アカルボースが1μM〜10μMで含まれる、[７]に記載の方法。
[１２]前記（ii）における基質液のpHが3.5〜4.5である、[７]又は[１１]に記載の方法。
[１３]前記（ii）における基質液が、クエン酸-リン酸ナトリウム緩衝液である、[７]、[１１]又は[１２]に記載の方法。
[１４]前記（iii）における4-Methyumbelliferyl合成基質が、4-methylum-belliferyl-β-D-glucopyranosideであり、1ｍM〜10ｍMで含まれる、[７]に記載の方法。
[１５]前記（iii）における基質液のpHが4.5〜5.5である、[７]又は[１４]に記載の方法。
[１６]前記（iii）における基質液が、クエン酸-リン酸ナトリウム緩衝液である、[７]、[１４]又は[１５]に記載の方法。
[１７]前記（iii）における基質液に、タウロデオキシコール酸ナトリウムが類似酵素阻害剤として含まれる、[７]及び[１４]から[１６]のいずれか一項に記載の方法。
[１８]工程（２）において、酵素反応の停止を、酵素反応溶液にグリシンを含む共通反応停止液を添加することにより行う、[１]から[１７]のいずれか一項に記載の方法。
[１９]前記共通反応停止液のpHが10〜11である、[１８]に記載の方法。
[２０]前記酵素反応を、25〜45℃の温度で行った後、前記共通反応停止液を添加する、[１８]又は[１９]に記載の方法。
[２１]前記酵素反応を2〜4時間で行う、[１]から[２０]のいずれか一項に記載の方法。
[２２]前記蛍光測定の測定波長が、励起波長365〜375nm、検出波長460〜470nmである、[１]から[２１]のいずれか一項に記載の方法。
[２３]１枚の乾燥血液ろ紙から複数のライソゾーム病責任酵素を同時に抽出する方法であって、共通抽出液を用いることを含み、該責任酵素がα-D-galactosidase A（GLA）、酸性α-glucosidase（GAA）及び酸性β-glucosidase（GBA）からなる群より選ばれる２以上の酵素である、方法。
[２４]前記共通抽出液のpHが5.5〜6.5である、[２３]に記載の方法。
[２５]前記共通抽出液が、リン酸イオン及びクエン酸イオンを含む緩衝液である、[２４]に記載の方法。
[２６]前記共通抽出液に、0.1ｍM〜5ｍMのDTTが含まれる、[２３]から[２５]のいずれか一項に記載の方法。
[２７]前記共通抽出液に、マグネシウムイオン（Mg2+）が含まれる、[２３]から[２６]のいずれか一項に記載の方法。
[２８]前記共通抽出液に、0.05％〜0.5％のTritonX-100及び0.02％〜0.1％のアジ化ナトリウムが含まれる、[２３]から[２７]のいずれか一項に記載の方法。
[２９]下記を含む、[１]に記載の検査方法に使用するためのキット：
（１）乾燥血液ろ紙、
（２）共通抽出液、
（３）合成基質を含む基質液、
（４）共通反応停止液、及び
（５）酵素反応により得られた生成物を蛍光測定する手段。
[３０]前記共通抽出液が、0.1％TrtionX-100、5mM MgCl2、0.5ｍM DTT及び0.05％アジ化ナトリウムを含むpH6.0の25ｍMクエン酸-25ｍMリン酸カリウム緩衝液である、[２９]に記載のキット。
[３１]前記合成基質を含む基質液が、ファブリー病を検出するためのGLA酵素活性測定用の基質液、ポンペ病を検出するためのGAA酵素活性測定用の基質液及びゴーシェ病を検出するためのGBA酵素活性測定用の基質液の組み合わせであり、ファブリー病を検出するためのGLA酵素活性測定用の基質液が、3.0ｍM 4-methylumbelliferyl-α-D-garactopyranoside及び100ｍM N-アセチル-D-ガラクトサミンを含むpH4.4の100ｍMクエン酸-200ｍMリン酸ナトリウム緩衝液、ポンペ病を検出するためのGAA酵素活性測定用の基質液が、2.0ｍM 4-methylumbelliferyl-α-D-glucopyranoside及び4.5μMアカルボースを含むpH4.0の250ｍMクエン酸-250ｍMリン酸カリウム緩衝液、ゴーシェ病を検出するためのGBA酵素活性測定用の基質液が、3.0ｍM 4-methylumbelliferyl-β-D−glucopyranoside及び0.3％タウロデオシキコール酸ナトリウムを含むpH5.0の100ｍMクエン酸-100ｍMリン酸ナトリウム緩衝液である、[２９]又は[３０]に記載のキット。
[３２]前記共通反応停止液が、300ｍMグリシン-NaOH緩衝液（pH10.6）である、[２９]から[３１]のいずれか一項に記載のキット。
Accordingly, the present invention includes the following.
[1] A rapid mass screening method for lysosomal disease responsible enzyme, which rapidly measures the activity of a plurality of lysosomal disease responsible enzymes from a single dry blood filter paper, wherein the responsible enzyme is α-D-galactosidase A (GLA ), An enzyme selected from the group consisting of acidic α-glucosidase (GAA) and acidic β-glucosidase (GBA), the method comprising the following steps:
(1) a step of extracting a blood sample from a dry blood filter paper using a common extract,
(2) adding a substrate solution containing a synthetic substrate to the extracted blood sample to cause an enzyme reaction;
(3) A step of measuring fluorescence of the product obtained by the enzyme reaction.
[2] The method according to [1], wherein the common extract has a pH of 5.5 to 6.5.
[3] The method according to [2], wherein the common extract is a buffer solution containing phosphate ions and citrate ions.
[4] The method according to any one of [1] to [3], wherein the common extract contains 0.1 mM to 5 mM of Dithiothreitol (DTT).
[5] The method according to any one of [1] to [4], wherein the common extract contains magnesium ions (Mg 2+ ).
[6] The method according to any one of [1] to [5], wherein the common extract contains 0.05% to 0.5% TritonX-100 and 0.02% to 0.1% sodium azide.
[7] The substrate solution comprises (i) a buffer solution containing N-acetylgalactosamine and 4-Methylumbelliferyl synthetic substrate, (ii) a buffer solution containing acarbose and 4-Methylumbelliferyl synthetic substrate, and (iii) 4-Methylumbelliferyl synthetic substrate. The method according to any one of [1] to [6], which is a combination of buffer solutions.
[8] The method according to [7], wherein the 4-Methyumbelliferyl synthetic substrate in (i) is 4-methylum-belliferyl-α-D-galactopyranoside, and is contained at 1 mM to 10 mM.
[9] The method according to [7] or [8], wherein the pH of the substrate solution in (i) is 4.0 to 5.0.
[10] The method according to any one of [7] to [9], wherein the substrate solution in (i) is a citrate-sodium phosphate buffer.
[11] The synthetic substrate of 4-Methyumbelliferyl in (ii) is 4-methylum-belliferyl-α-D-glucopyranoside, contained at 1 mM to 10 mM, and acarbose is contained at 1 μM to 10 μM. the method of.
[12] The method according to [7] or [11], wherein the pH of the substrate solution in (ii) is 3.5 to 4.5.
[13] The method according to [7], [11] or [12], wherein the substrate solution in (ii) is a citrate-sodium phosphate buffer.
[14] The method according to [7], wherein the 4-Methyumbelliferyl synthetic substrate in (iii) is 4-methylum-belliferyl-β-D-glucopyranoside, and is contained at 1 mM to 10 mM.
[15] The method according to [7] or [14], wherein the pH of the substrate solution in (iii) is 4.5 to 5.5.
[16] The method according to [7], [14] or [15], wherein the substrate solution in (iii) is a citrate-sodium phosphate buffer.
[17] The method according to any one of [7] and [14] to [16], wherein the substrate solution in (iii) contains sodium taurodeoxycholate as a similar enzyme inhibitor.
[18] The method according to any one of [1] to [17], wherein in step (2), the enzyme reaction is stopped by adding a common reaction stop solution containing glycine to the enzyme reaction solution.
[19] The method according to [18], wherein the common reaction stop solution has a pH of 10 to 11.
[20] The method according to [18] or [19], wherein the enzyme reaction is performed at a temperature of 25 to 45 ° C., and then the common reaction stop solution is added.
[21] The method according to any one of [1] to [20], wherein the enzyme reaction is performed in 2 to 4 hours.
[22] The method according to any one of [1] to [21], wherein a measurement wavelength of the fluorescence measurement is an excitation wavelength of 365 to 375 nm and a detection wavelength of 460 to 470 nm.
[23] A method for simultaneously extracting a plurality of lysosomal disease responsible enzymes from a sheet of dried blood filter paper, comprising using a common extract, wherein the responsible enzyme is α-D-galactosidase A (GLA), acidic α A method, which is two or more enzymes selected from the group consisting of -glucosidase (GAA) and acidic β-glucosidase (GBA).
[24] The method according to [23], wherein the common extract has a pH of 5.5 to 6.5.
[25] The method according to [24], wherein the common extract is a buffer containing phosphate ions and citrate ions.
[26] The method according to any one of [23] to [25], wherein the common extract contains 0.1 mM to 5 mM DTT.
[27] The method according to any one of [23] to [26], wherein the common extract contains magnesium ions (Mg 2+ ).
[28] The method according to any one of [23] to [27], wherein the common extract contains 0.05% to 0.5% TritonX-100 and 0.02% to 0.1% sodium azide.
[29] A kit for use in the inspection method according to [1], comprising:
(1) dry blood filter paper,
(2) common extract,
(3) a substrate solution containing a synthetic substrate,
(4) Common reaction stop solution, and (5) Means for fluorescence measurement of the product obtained by the enzyme reaction.
[30] The common extract is a 25 mM citrate-25 mM potassium phosphate buffer, pH 6.0, containing 0.1% TrtionX-100, 5 mM MgCl 2 , 0.5 mM DTT and 0.05% sodium azide. [29] The kit according to 1.
[31] For detecting a substrate solution for measuring GLA enzyme activity for detecting Fabry disease, a substrate solution for measuring GAA enzyme activity for detecting Pompe disease, and Gaucher disease, wherein the substrate solution containing the synthetic substrate is Is a combination of a substrate solution for measuring GBA enzyme activity and a substrate solution for measuring GLA enzyme activity for detecting Fabry disease is 3.0 mM 4-methylumbelliferyl-α-D-garactopyranoside and 100 mM N-acetyl-D- A pH 4.4 100 mM citrate-200 mM sodium phosphate buffer containing galactosamine, a substrate solution for measuring GAA enzyme activity to detect Pompe disease, 2.0 mM 4-methylumbelliferyl-α-D-glucopyranoside and 4.5 μM acarbose PH 4.0 250 mM citrate-250 mM potassium phosphate buffer, GBA enzyme activity substrate solution for detecting Gaucher disease is 3.0 mM 4-methylumbelliferyl-β-D-glucopyranoside and 0.3% taurodeo Succiolate A 100mM citrate -100mM sodium phosphate buffer pH5.0 containing um, kit according to [29] or [30].
[32] The kit according to any one of [29] to [31], wherein the common reaction stop solution is a 300 mM glycine-NaOH buffer (pH 10.6).
本発明は、乾燥血液ろ紙の枚数が制限される新生児マススクリーニング検査において、１枚の乾燥血液ろ紙から、ライソゾーム病３疾患（ファブリー病、ポンペ病、ゴーシェ病）の酵素活性測定を可能とした。 The present invention makes it possible to measure enzyme activity of three lysosomal diseases (Fabry disease, Pompe disease, Gaucher disease) from one dry blood filter paper in a newborn mass screening test in which the number of dry blood filter papers is limited.
本発明は、測定条件を共通化させたことで、多数の検体を一度に測定可能とし、検査に要する時間を大幅に短縮させ、これまで２日間要していた検査期間を１日で完了させることを可能とした。さらに、測定者の負担の軽減（省力化）を成し遂げた。 The present invention makes it possible to measure a large number of specimens at the same time by making the measurement conditions common, greatly reducing the time required for the test, and completing the test period which has been required for two days in one day. Made it possible. In addition, the burden on the measurer was reduced (labor saving).
本発明により、検体としての血液が採取しにくい新生児について、既存の新生児代謝異常で使用した乾燥血液ろ紙をそのまま使用することができるようになった。すべての新生児について、３種のライソゾーム病の可能性が検出できれば、未だ臨床症状が現れていない出生後の早期の段階で、酵素補充療法、遺伝子治療、骨髄移植等が実施でき、精神発達の遅れ等をくい止めることができる可能性が高くなる。 According to the present invention, it is possible to use a dry blood filter paper that has been used for neonatal metabolic abnormalities as it is for newborns from which it is difficult to collect blood as a specimen. For all newborns, if the possibility of three types of lysosomal disease can be detected, enzyme replacement therapy, gene therapy, bone marrow transplantation, etc. can be performed at an early stage after birth where clinical symptoms have not yet appeared, and mental development is delayed There is a high possibility that it can be stopped.
さらに本発明は、マススクリーニング検査でのライソゾーム病の検出のみならず、病状の把握、治療方針の決定、治療効果の確認、経過観察、モニタリング、医薬品開発の評価等へも応用することができる。 Furthermore, the present invention can be applied not only to detection of lysosomal disease in mass screening tests, but also to grasping disease states, determining treatment policies, confirming therapeutic effects, follow-up observation, monitoring, evaluation of drug development, and the like.
（発明の詳細な説明）
乾燥血液ろ紙を検体としたライソゾーム病責任酵素活性測定は、乾燥血液ろ紙と基質液を直接混合して酵素反応を行わせる方法や、乾燥血液ろ紙に少量のbufferを添加した後、基質液を添加して反応させる方法、乾燥血液ろ紙から血液を抽出して、抽出した血液試料に基質液を添加して酵素反応を行わせる方法などがある。
(Detailed description of the invention)
Lysozyme disease responsible enzyme activity measurement using dry blood filter paper as a sample is a method of directly mixing dry blood filter paper and substrate solution to perform an enzyme reaction, or adding a small amount of buffer to dry blood filter paper and then adding substrate solution And a method of extracting blood from a dry blood filter paper and adding a substrate solution to the extracted blood sample to cause an enzyme reaction.
本発明は、１枚の乾燥血液ろ紙から、ファブリー病責任酵素（α-D-galactosidase A: 以下、「GLA」と略す。)、ポンペ病責任酵素（酸性α-glucosidase：以下、「GAA」と略す。）及びゴーシェ病責任酵素（酸性β-glucosidase：以下「GBA」と略す。）を含む血液を抽出し、抽出した血液試料を利用して、乾燥血液ろ紙中の３種の酵素活性を測定する方法である。 In the present invention, Fabry disease responsible enzyme (α-D-galactosidase A: hereinafter abbreviated as “GLA”), Pompe disease responsible enzyme (acid α-glucosidase: hereinafter referred to as “GAA”) from a single dried blood filter paper. And blood containing Gaucher disease responsible enzyme (acid β-glucosidase: hereinafter referred to as “GBA”) is extracted, and three enzyme activities in dry blood filter paper are measured using the extracted blood sample. It is a method to do.
本発明方法による測定は、（１）乾燥血液ろ紙から共通抽出液を用いて血液試料を抽出する工程、（２）抽出した血液試料に合成基質を添加して、酵素反応を行わせる工程、（３）酵素反応により得られた生成物を蛍光測定する工程の３ステップにより完了する。 The measurement according to the method of the present invention includes (1) a step of extracting a blood sample from a dry blood filter paper using a common extract, (2) a step of adding a synthetic substrate to the extracted blood sample and causing an enzymatic reaction to be performed, ( 3) It is completed in 3 steps of the process of measuring the fluorescence of the product obtained by the enzyme reaction.
血液試料の酵素活性測定では、抽出した血液試料と基質を混合した場合に酵素活性測定に適するpHになるように、抽出液と基質液のどちらか一方で、又は両方で最適pHの緩衝能を持つ組成にする必要があり、測定する酵素毎に抽出液が異なっていた。このため、測定する酵素毎に別々の乾燥血液ろ紙が必要であった。 When measuring the enzyme activity of a blood sample, when the extracted blood sample and the substrate are mixed, the buffer pH of the optimum pH is set in either the extract solution or the substrate solution, or both so that the pH is suitable for enzyme activity measurement. It was necessary to have a composition, and the extract was different for each enzyme to be measured. For this reason, a separate dry blood filter paper was required for each enzyme to be measured.
本発明では、GLA、GAA、及びGBAの酵素活性測定を妨害せず、かつ乾燥血液ろ紙から効果的に各酵素を同時に抽出することができる共通抽出液組成を見出し、１枚の乾燥血液ろ紙から複数の酵素を同時に測定する方法を可能とした。 In the present invention, a common extract composition that can simultaneously extract each enzyme from a dry blood filter paper is found without interfering with the enzyme activity measurement of GLA, GAA, and GBA, and from one dry blood filter paper. A method for simultaneously measuring a plurality of enzymes has been made possible.
本発明方法による共通抽出液は、各基質液と混合したときに酵素反応の最適pHを阻害しない低緩衝能の緩衝液が好ましく、より好ましくは、基質液の緩衝能の1/10程度の緩衝能を持つ緩衝液である。共通抽出液のpHは、5.5〜6.5が好ましく、6.0がより好ましい。 The common extract according to the method of the present invention is preferably a buffer solution having a low buffer capacity that does not inhibit the optimum pH of the enzyme reaction when mixed with each substrate solution, and more preferably a buffer having about 1/10 of the buffer capacity of the substrate solution. It is a buffer solution with ability. The pH of the common extract is preferably 5.5 to 6.5, more preferably 6.0.
本発明方法による共通抽出液は、空気中の酸素による酸化を受けた乾燥血液ろ紙が、抽出でさらなる酸化を受けない組成であることが好まれる。よって、弱酸性域に緩衝能を持つ還元作用のあるクエン酸-リン酸緩衝液が好ましい。また、測定する酵素は、白血球などの血液細胞中のライソゾーム中に存在するため、細胞を破砕し、抽出液中に酵素を遊離させる作用を持つ界面活性剤（TritonX-100など）を共存させることがより好ましい。 The common extract obtained by the method of the present invention preferably has a composition in which the dried blood filter paper that has been oxidized by oxygen in the air does not undergo further oxidation upon extraction. Therefore, a citrate-phosphate buffer solution having a reducing action having a buffer capacity in a weakly acidic region is preferable. In addition, since the enzyme to be measured is present in lysosomes in blood cells such as leukocytes, a surfactant (such as TritonX-100) that disrupts the cells and releases the enzyme in the extract must be present. Is more preferable.
本発明による共通抽出液は、測定対象の酵素活性の抑制には働かず、酵素を抽出する過程で目的酵素の活性増強作用を持つ添加物を共存させることで、抽出時の酵素の失活を抑制する作用を持ち、かつ乾燥血液ろ紙からの抽出を促進させる界面活性剤を共存させることが好ましい。界面活性剤は、一般的に作用させる濃度では酵素反応を抑制したり、増強させたりすることがあるため、酵素活性を阻害しない濃度で添加する必要がある。 The common extract according to the present invention does not act on the inhibition of the enzyme activity to be measured, and coexists with an additive having an activity enhancing activity of the target enzyme in the process of extracting the enzyme, thereby inactivating the enzyme during extraction. It is preferable to coexist with a surfactant that has an inhibitory action and promotes extraction from dry blood filter paper. A surfactant may suppress or enhance an enzyme reaction at a concentration at which it generally acts, so it is necessary to add the surfactant at a concentration that does not inhibit the enzyme activity.
本発明の共通抽出液は、0.05％〜0.5％のTrtionX-100、1mM〜10mMのMgCl2、0.1mM〜5mMのDithiothreitol（以下、「DTT」と略す。）及び0.02％〜0.1％のアジ化ナトリウムを含む。本発明の一実施形態の共通抽出液の組成は、「0.1％ TrtionX-100、5mM MgCl2、0.5mM DTT、0.05％アジ化ナトリウムを含むpH6.0の25ｍMクエン酸-25ｍMリン酸カリウム緩衝液」である。共通抽出液中のDTTは、GLA及びGAAの酵素反応には影響せず、GBAの酵素活性を増強する。また、MgCl2より提供されるMg2+イオンは、GLAの増強に作用し、リン酸カリウムより提供されるK+イオンはGAAの酵素反応を増強する作用がある。界面活性剤であるTritonX-100は、乾燥血液ろ紙からの血液成分の溶出と血球破砕による酵素の溶出を促進し、抽出時間の短縮のために添加する。各添加剤の濃度は、互いの酵素活性を抑制しない濃度として設定することが好ましい。 The common extract of the present invention comprises 0.05% to 0.5% TrtionX-100, 1 mM to 10 mM MgCl 2 , 0.1 mM to 5 mM Dithiothreitol (hereinafter abbreviated as “DTT”) and 0.02% to 0.1% azide. Contains sodium. The composition of the common extract of one embodiment of the present invention is “25 mM citrate-25 mM potassium phosphate buffer solution of pH 6.0 containing 0.1% TrtionX-100, 5 mM MgCl 2 , 0.5 mM DTT, 0.05% sodium azide. Is. DTT in the common extract does not affect the enzymatic reaction of GLA and GAA, but enhances the enzymatic activity of GBA. Further, Mg 2+ ions provided from MgCl 2 act to enhance GLA, and K + ions provided from potassium phosphate have an action to enhance the enzymatic reaction of GAA. TritonX-100, a surfactant, is added to accelerate the elution of blood components from dry blood filter paper and the elution of enzymes by crushing blood cells, and shorten the extraction time. The concentration of each additive is preferably set as a concentration that does not inhibit the enzyme activity of each other.
本発明による基質液は、それぞれの酵素活性測定に適したpH域で緩衝能を持つ緩衝液を使用することにより、共通抽出液で抽出した血液試料を用いて各酵素の測定が可能となる。 The substrate solution according to the present invention can measure each enzyme using a blood sample extracted with a common extract by using a buffer solution having a buffer capacity in a pH range suitable for each enzyme activity measurement.
基質液に使用する合成基質は、測定対象酵素の特異基質となる構造を有する基質であることが必要である。そのような合成基質を使用することにより、測定対象酵素との反応特異性を向上させることができる。しかしながら、抽出された血液試料中には、類似反応をする酵素が数多く存在するため、さらに特異性を高める目的で、類似酵素阻害剤を共存させることが好ましい。 The synthetic substrate used in the substrate solution needs to be a substrate having a structure that becomes a specific substrate of the enzyme to be measured. By using such a synthetic substrate, the reaction specificity with the enzyme to be measured can be improved. However, in the extracted blood sample, there are many enzymes that react similarly, and therefore, it is preferable that a similar enzyme inhibitor coexists for the purpose of further increasing the specificity.
ファブリー病を検出するためのGLA酵素活性測定に使用する基質液は、1mM〜10mMの4-methylumbelliferyl-α-D-garactopyranoside及び10mM〜200mMのN-アセチル-D-ガラクトサミンを含む。ファブリー病を検出するためのGLA酵素活性測定に使用する基質液の一実施形態における組成は、「3.0ｍM 4-methylumbelliferyl-α-D-garactopyranoside及び100ｍM N-アセチル-D-ガラクトサミンを含むpH4.4の100ｍMクエン酸-200ｍMリン酸ナトリウム緩衝液」である。pHは、4.0〜5.0であっても良い。 The substrate solution used for measuring the GLA enzyme activity for detecting Fabry disease contains 1 mM to 10 mM 4-methylumbelliferyl-α-D-garactopyranoside and 10 mM to 200 mM N-acetyl-D-galactosamine. The composition in one embodiment of the substrate solution used for measuring the GLA enzyme activity for detecting Fabry disease is “pH 4.4 containing 3.0 mM 4-methylumbelliferyl-α-D-garactopyranoside and 100 mM N-acetyl-D-galactosamine”. Of 100 mM citrate-200 mM sodium phosphate buffer ". The pH may be 4.0 to 5.0.
ポンペ病を検出するためのGAA酵素活性測定に使用する基質液は、1mM〜10mMの4-methylumbelliferyl-α-D-glucopyranoside及び1μM〜10μMのアカルボースを含む。ポンペ病を検出するためのGAA酵素活性測定に使用する基質液の一実施形態における組成は、「2.0ｍM 4-methylumbelliferyl-α-D-glucopyranoside、4.5μMアカルボースを含むpH4.0の250ｍMクエン酸-250ｍMリン酸カリウム緩衝液」である。pHは、3.5〜4.5であっても良い。アカルボースは、血液試料中に含まれるGAA以外のα-glucosidaseの作用を阻害させるために添加する。 The substrate solution used for GAA enzyme activity measurement for detecting Pompe disease contains 1 mM to 10 mM 4-methylumbelliferyl-α-D-glucopyranoside and 1 μM to 10 μM acarbose. The composition of an embodiment of a substrate solution used for measuring GAA enzyme activity for detecting Pompe disease is “250 mM citric acid at pH 4.0 containing 2.0 mM 4-methylumbelliferyl-α-D-glucopyranoside, 4.5 μM acarbose— "250 mM potassium phosphate buffer". The pH may be 3.5 to 4.5. Acarbose is added to inhibit the action of α-glucosidase other than GAA contained in the blood sample.
ゴーシェ病を検出するためのGBA酵素活性測定に使用する基質液は、1mM〜10mMの4-methylumbelliferyl-β-D−glucopyranoside及び0.1％〜1％のタウロデオシキコール酸ナトリウムを含む。ゴーシェ病を検出するためのGBA酵素活性測定に使用する基質液の一実施形態における組成は、「3.0ｍM 4-methylumbelliferyl-β-D−glucopyranoside、0.3％タウロデオシキコール酸ナトリウムを含むpH5.0の100ｍMクエン酸-100ｍMリン酸ナトリウム緩衝液」である。pHは、4.5〜5.5であっても良い。 The substrate solution used for GBA enzyme activity measurement for detecting Gaucher disease contains 1 mM to 10 mM 4-methylumbelliferyl-β-D-glucopyranoside and 0.1% to 1% sodium taurodeoxycholate. The composition of one embodiment of the substrate solution used for GBA enzyme activity measurement for detecting Gaucher disease is “3.0 mM 4-methylumbelliferyl-β-D-glucopyranoside, pH 5.0 containing 0.3% sodium taurodeoxycholate. "100 mM citrate-100 mM sodium phosphate buffer". The pH may be 4.5 to 5.5.
ゴーシェ病用基質液に添加される類似酵素阻害剤は、タウロコール酸ナトリウムが一般的に使用されているが、タウロコール酸ナトリウムよりもGBA酵素活性増強に効果があるタウロデオキシコール酸ナトリウムを使用することで、低濃度の試料のGBA活性が測定可能となる。 As a similar enzyme inhibitor added to Gaucher's disease substrate solution, sodium taurocholate is generally used, but sodium taurodeoxycholate, which is more effective in enhancing GBA enzyme activity than sodium taurocholate, should be used. Thus, the GBA activity of a low concentration sample can be measured.
各酵素反応により生成する共通の反応生成物は、アルカリ域に強い蛍光を発する4-methylumbelliferone（以下「4MU」と略す。）であり、この4MUの蛍光強度を測定する。4MUは、pH９〜11のアルカリ域で最も強い蛍光を発するため、共通反応停止液を添加した後の反応液のpHをアルカリ側にシフトさせることが好ましい。しかしながら、各酵素反応においてpHが異なること、及び基質液はアルカリ域に強い緩衝能を持つことから、共通反応停止液は、それらの影響を受けないよう、高濃度のアルカリ緩衝液とすることがより好ましい。 The common reaction product produced by each enzyme reaction is 4-methylumbelliferone (hereinafter abbreviated as “4MU”) that emits strong fluorescence in the alkaline region, and the fluorescence intensity of this 4MU is measured. Since 4MU emits the strongest fluorescence in the alkaline region of pH 9 to 11, it is preferable to shift the pH of the reaction solution after adding the common reaction stop solution to the alkali side. However, since each enzyme reaction has a different pH and the substrate solution has a strong buffer capacity in the alkaline region, the common reaction stop solution should be a high-concentration alkaline buffer solution so as not to be affected by them. More preferred.
本発明における共通反応停止液は、100ｍM〜500ｍMのグリシン-NaOH緩衝液（pH8〜11）である。この共通反応停止液組成を使用することにより、酵素反応を停止させると共に反応生成物（4MU）の蛍光を増強させて測定することができる。4MUは、pH8を超えるアルカリ域では、360nm付近の紫外線を吸収して、450nm付近に最大蛍光強度を持つ蛍光を発する。よって、共通反応停止液のpHは、8以上が好ましく、より好ましくは9以上であり、10〜11がさらに好ましい。本発明の一実施形態で使用可能な共通反応停止液の組成は、最も好ましくは「300ｍMグリシン-NaOH緩衝液（pH10.6）」である。 The common reaction stop solution in the present invention is 100 mM to 500 mM glycine-NaOH buffer (pH 8 to 11). By using this common reaction stop solution composition, the enzyme reaction can be stopped and the fluorescence of the reaction product (4MU) can be enhanced. 4MU absorbs ultraviolet light around 360nm in the alkaline region above pH 8, and emits fluorescence with maximum fluorescence intensity around 450nm. Therefore, the pH of the common reaction stop solution is preferably 8 or more, more preferably 9 or more, and further preferably 10 to 11. The composition of the common reaction stop solution that can be used in one embodiment of the present invention is most preferably “300 mM glycine-NaOH buffer (pH 10.6)”.
乾燥血液ろ紙抽出物には、ヘモグロビンなどの4MU蛍光測定を妨害する色素蛋白や、紫外線を吸収して4MU励起効率を阻害する核酸成分などが含まれる。本発明では、血液成分を含む反応液での測定で、血液成分の阻害を少なくした状態で4MUを測定するために、測定の波長を4MUの最大励起と最大蛍光波長を意図的にシフトさせて測定する。 The dried blood filter paper extract contains a chromoprotein that interferes with 4MU fluorescence measurement, such as hemoglobin, and a nucleic acid component that absorbs ultraviolet light and inhibits 4MU excitation efficiency. In the present invention, the measurement wavelength is intentionally shifted between the maximum excitation of the 4MU and the maximum fluorescence wavelength in order to measure 4MU in a state where the inhibition of the blood component is reduced in the measurement with the reaction solution containing the blood component. taking measurement.
この励起波長と測定波長のシフトにより、これまで蛍光を測定する前に必要であった遠心による蛍光阻害物質の分離工程と測定用プレートへの試料の移注作業を削減することができ、測定操作の簡便性が向上した。 This shift between the excitation wavelength and the measurement wavelength can reduce the separation process of fluorescence inhibitory substances by centrifugation and the work of transferring the sample to the measurement plate, which were necessary before measuring fluorescence. Improved convenience.
本発明での一実施形態での測定波長は、励起波長365〜375nmと検出波長460〜470nmの組合せであり、より好ましくは、励起波長370nｍと検出波長465nmの組み合わせである。この測定波長の組み合わせを各酵素測定共通で使用することにより、１台の蛍光リーダーで各酵素活性を同一マイクロプレート上に配置した場合でも測定可能となった。 In one embodiment of the present invention, the measurement wavelength is a combination of an excitation wavelength of 365 to 375 nm and a detection wavelength of 460 to 470 nm, and more preferably a combination of an excitation wavelength of 370 nm and a detection wavelength of 465 nm. By using this combination of measurement wavelengths in common with each enzyme measurement, it becomes possible to measure even when each enzyme activity is arranged on the same microplate with one fluorescence reader.
酵素活性は、乾燥血液ろ紙１枚当たりの単位時間に得られる4MU産生量として計算する。4MU産生量は、4MU標準品の蛍光強度と抽出試料の蛍光強度を比較して算出する。また、酵素活性は、以下の計算式により算出する。
酵素活性（pmol/hr/disk)＝抽出試料の4MU量(μM)×抽出試料量（μL）÷酵素反応時間（hr）×（乾燥血液ろ紙１枚当たりの抽出液量（μL）÷抽出試料量（μL））
Enzyme activity is calculated as the amount of 4MU produced per unit time per sheet of dry blood filter paper. The amount of 4MU produced is calculated by comparing the fluorescence intensity of the 4MU standard with the fluorescence intensity of the extracted sample. The enzyme activity is calculated by the following formula.
Enzyme activity (pmol / hr / disk) = 4MU amount of extracted sample (μM) x Extracted sample amount (μL) ÷ Enzyme reaction time (hr) x (Extract volume per dry blood filter paper (μL) ÷ Extracted sample Volume (μL))
本発明での一実施形態での測定操作は、乾燥血液ろ紙１枚をマイクロプレートのwell内に入れ、100μLの共通抽出液を添加して、１時間の抽出反応を行わせる。抽出された血液試料は、20μLずつ別のマイクロプレートにwell to wellで移注し、そこに各基質液を40μL添加して、25℃〜45℃で2時間〜24時間反応させる。 In the measurement operation according to an embodiment of the present invention, one dry blood filter paper is put into a well of a microplate, 100 μL of a common extract is added, and an extraction reaction is performed for 1 hour. 20 μL of the extracted blood sample is transferred to another microplate in a well-to-well manner, 40 μL of each substrate solution is added thereto, and reacted at 25 to 45 ° C. for 2 to 24 hours.
酵素反応温度は、３種の責任酵素で挙動が異なる。GAAとGBAは37℃付近、GAAは45℃付近が最も活性が高い。よって、共通で使用する温度条件としては、35〜45℃が好ましく、より好ましくは37℃付近である。 The enzyme reaction temperature behaves differently among the three responsible enzymes. GAA and GBA are most active around 37 ° C, and GAA is most active around 45 ° C. Accordingly, the temperature condition commonly used is preferably 35 to 45 ° C, more preferably around 37 ° C.
酵素反応時間は一般的に24時間であるが、測定酵素により挙動が異なるため、本測定系の場合、2〜4時間での酵素活性が、24時間反応させた場合より高活性を示す。よって、共通で使用する条件としては、2〜4時間が好ましい。酵素反応終了後、各反応液には、共通反応停止液を200μL添加して酵素反応を停止させる。蛍光マイクロプレートリーダーを用いて、励起波長370nm、検出波長465nmで蛍光を測定し、4MU標準品の蛍光強度と比較して酵素活性を算出する。 The enzyme reaction time is generally 24 hours, but the behavior differs depending on the enzyme to be measured. Therefore, in the case of this measurement system, the enzyme activity in 2 to 4 hours shows a higher activity than in the case of reacting for 24 hours. Therefore, 2 to 4 hours are preferable as the conditions used in common. After completion of the enzyme reaction, 200 μL of a common reaction stop solution is added to each reaction solution to stop the enzyme reaction. Fluorescence is measured at an excitation wavelength of 370 nm and a detection wavelength of 465 nm using a fluorescence microplate reader, and the enzyme activity is calculated by comparison with the fluorescence intensity of the 4MU standard product.
本発明はまた、本発明の検査方法に使用するためのキットを提供する。本発明のキットは、以下の構成を含む：
（１）乾燥血液ろ紙、
（２）共通抽出液、
（３）合成基質を含む基質液、
（４）共通反応停止液、及び
（５）酵素反応により得られた生成物を蛍光測定する手段。
The present invention also provides a kit for use in the inspection method of the present invention. The kit of the present invention comprises the following configuration:
(1) dry blood filter paper,
(2) common extract,
(3) a substrate solution containing a synthetic substrate,
(4) Common reaction stop solution, and (5) Means for fluorescence measurement of the product obtained by the enzyme reaction.
本発明のキットに含まれる前記共通抽出液は、最も好ましくは、0.1％TrtionX-100、5ｍM MgCl2、0.5ｍM DTT及び0.05％アジ化ナトリウムを含む。 The common extract contained in the kit of the present invention most preferably contains 0.1% TrtionX-100, 5 mM MgCl 2 , 0.5 mM DTT and 0.05% sodium azide.
本発明のキットに含まれる前記合成基質を含む基質液は、ファブリー病を検出するためのGLA酵素活性測定用の基質液、ポンペ病を検出するためのGAA酵素活性測定用の基質液及びゴーシェ病を検出するためのGBA酵素活性測定用の基質液の組み合わせであり、最も好ましくは、ファブリー病を検出するためのGLA酵素活性測定用の基質液が、3.0ｍM 4-methylumbelliferyl-α-D-garactopyranoside及び100ｍM N-アセチル-D-ガラクトサミンを含むpH4.4の100ｍMクエン酸-200ｍMリン酸ナトリウム緩衝液であり、ポンペ病を検出するためのGAA酵素活性測定用の基質液が、2.0ｍM 4-methylumbelliferyl-α-D-glucopyranoside及び4.5μMアカルボースを含むpH4.0の250ｍMクエン酸-250ｍMリン酸カリウム緩衝液であり、ゴーシェ病を検出するためのGBA酵素活性測定用の基質液が、3.0ｍM 4-methylumbelliferyl-β-D−glucopyranoside及び0.3％タウロデオシキコール酸ナトリウムを含むpH5.0の100ｍMクエン酸-100ｍMリン酸ナトリウム緩衝液である。 The substrate solution containing the synthetic substrate contained in the kit of the present invention includes a substrate solution for measuring GLA enzyme activity for detecting Fabry disease, a substrate solution for measuring GAA enzyme activity for detecting Pompe disease, and Gaucher disease. Is a combination of substrate solutions for measuring GBA enzyme activity for detecting GLA enzyme activity, and most preferably, the substrate solution for measuring GLA enzyme activity for detecting Fabry disease is 3.0 mM 4-methylumbelliferyl-α-D-garactopyranoside And a 100 mM citrate-200 mM sodium phosphate buffer solution with pH 4.4 containing 100 mM N-acetyl-D-galactosamine, and a substrate solution for measuring GAA enzyme activity for detecting Pompe disease is 2.0 mM 4-methylumbelliferyl -α-D-glucopyranoside and 4.5 μM acarbose pH 4.0 250 mM citrate-250 mM potassium phosphate buffer, and a substrate solution for measuring GBA enzyme activity for detecting Gaucher disease is 3.0 mM 4- methylumbelliferyl-β-D A glucopyranoside, and 100mM citrate -100mM sodium phosphate buffer pH5.0 containing 0.3% tau Rodeo sodium Shikikoru acid.
本発明のキットに含まれる前記共通反応停止液は、最も好ましくは、300ｍMグリシン-NaOH緩衝液（pH10.6）である。
以下、本発明を実施例により具体的に説明するが、本発明はこれらの実施例によって何ら限定されるものではない。
The common reaction stop solution contained in the kit of the present invention is most preferably 300 mM glycine-NaOH buffer (pH 10.6).
EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.
乾燥血液ろ紙を検体としてGLA、GAA、及びGBAの酵素活性を酵素反応の温度を変えて検出を試みた。
（１）検体、試薬等
本実施例において用いた検体、試薬等は以下のとおりである。
［検体及び標準品］
検体：正常人ヒト乾燥血液ろ紙（新生児72名の各乾燥血液ろ紙）、及びcontrol乾燥血液ろ紙（３種）
・4MU標準品：4-methylumbelliferone（SIGMA-ALDRICH社製、Product No.M1381）
Using dry blood filter paper as a specimen, the enzyme activities of GLA, GAA and GBA were detected by changing the temperature of the enzyme reaction.
(1) Sample, Reagent, etc. Samples, reagents, etc. used in this example are as follows.
[Samples and standard products]
Specimen: Normal human dry blood filter paper (each 72 newborn blood filter paper) and control dry blood filter paper (3 types)
・ 4MU standard product: 4-methylumbelliferone (manufactured by SIGMA-ALDRICH, Product No.M1381)
［合成基質］
・4-methylum-belliferyl-α-D-galactopyranoside（SIGMA-ALDRICHI社製、Product No. M7633）
・4-methylum-belliferyl-α-D-glucopyranoside（SIGMA-ALDRICHI社製、Product No. M9766）
・4-methylum-belliferyl-β-D-glucopyranoside（SIGMA-ALDRICHI社製、Product No. M3633）
[Synthetic substrate]
・ 4-methylum-belliferyl-α-D-galactopyranoside (manufactured by SIGMA-ALDRICHI, Product No. M7633)
・ 4-methylum-belliferyl-α-D-glucopyranoside (manufactured by SIGMA-ALDRICHI, Product No. M9766)
・ 4-methylum-belliferyl-β-D-glucopyranoside (SIGMA-ALDRICHI, Product No. M3633)
［酵素阻害剤］
・N-アセチル-D-ガラクトサミン：和光純薬社製（Product No. 013-1282）
・アカルボース：和光純薬社製（Product No. 019-22671）
・タウロデオキシコール酸ナトリウム（Sodium taurodeoxycholate hydrate）：SIGMA-ALDRICHI社製（Product No. T0557P）
[Enzyme inhibitor]
・ N-acetyl-D-galactosamine: Wako Pure Chemical Industries (Product No. 013-1282)
・ Acarbose: Wako Pure Chemical Industries, Ltd. (Product No. 019-22671)
・ Sodium taurodeoxycholate hydrate: SIGMA-ALDRICHI (Product No. T0557P)
共通抽出液は、以下の組成で調製した。
・共通抽出液：0.1％TrtionX-100、5mM MgCl2、0.5ｍM DTT及び0.05％アジ化ナトリウムを含むpH6.0の25ｍMクエン酸-25ｍMリン酸カリウム緩衝液
The common extract was prepared with the following composition.
· Common extract: 0.1% TrtionX-100,5mM MgCl 2 , 0.5mM DTT and 25mM citrate -25mM potassium phosphate buffer pH6.0 containing 0.05% sodium azide
各基質液は、以下の組成で調製した。
・ファブリー病用基質液：3.0ｍM 4-methylumbelliferyl-α-D-garactopyranoside及び100ｍM N-アセチル-D-ガラクトサミンを含むpH4.4の100ｍMクエン酸-200ｍMリン酸ナトリウム緩衝液
・ポンペ病用基質液：2.0ｍM 4-methylumbelliferyl-α-D-glucopyranoside及び4.5μMアカルボースを含むpH4.0の250ｍMクエン酸-250ｍMリン酸カリウム緩衝液
・ゴーシェ病用基質液：3.0ｍM 4-methylumbelliferyl-β-D−glucopyranoside及び0.3％タウロデオシキコール酸ナトリウムを含むpH5.0の100ｍMクエン酸-100ｍMリン酸ナトリウム緩衝液
Each substrate solution was prepared with the following composition.
・ Fabry disease substrate solution: 3.0 mM 4-methylumbelliferyl-α-D-garactopyranoside and 100 mM N-acetyl-D-galactosamine, pH 4.4 100 mM citrate-200 mM sodium phosphate buffer solution ・ Pompe disease substrate solution: PH 4.0 250 mM citrate-250 mM potassium phosphate buffer / Gaucher disease substrate solution containing 3.0 mM 4-methylumbelliferyl-α-D-glucopyranoside and 4.5 μM acarbose: 3.0 mM 4-methylumbelliferyl-β-D-glucopyranoside and 100 mM citrate-100 mM sodium phosphate buffer, pH 5.0, containing 0.3% sodium taurodeoxycholate
共通反応停止液は、以下の組成で調製した。
・共通反応停止液：300ｍMグリシン-NaOH緩衝液（pH10.6）
The common reaction stop solution was prepared with the following composition.
・ Common reaction stop solution: 300 mM glycine-NaOH buffer (pH 10.6)
（２）測定操作
本実施例における測定操作は以下のとおりである。
各乾燥血液ろ紙より、φ3.0mmの乾燥血液ろ紙検体を96wellマイクロプレートプレートの各wellに切り出し、各wellに100μLの共通抽出液を添加して、室温で振とうしながら1時間の抽出操作を行った。抽出された血液試料を20μLずつ３枚の蛍光測定用96well Blackマイクロプレート（NUNC社製）にwell to wellで移注した。4MU標準品は、2.5μMより2倍段階希釈した希釈標準液を血液試料と同様に20μL蛍光測定用96well Blackマイクロプレートに移注した。検体及び4MU標準液の入ったwellにそれぞれの基質を40μL添加し、25℃、37℃、45℃の各恒温槽内に4時間静置して酵素反応を行わせた。酵素反応終了後、共通反応停止液を各well 200μL添加して、軽く混合した後、蛍光オートリーダー（TECASN社製、infinite M200pro）を用いて、励起波長370nm、検出波長465nmで蛍光強度を測定し、4MU標準液の蛍光強度と比較して各乾燥血液ろ紙１枚当たりの酵素活性を求めた。
(2) Measurement operation The measurement operation in this example is as follows.
From each dried blood filter paper, cut a 3.0 mm diameter dried blood filter paper sample into each well of a 96-well microplate plate, add 100 μL of common extract to each well, and perform an extraction operation for 1 hour while shaking at room temperature. went. The extracted blood sample was transferred in a well-to-well manner to three 96-well Black microplates (manufactured by NUNC) for fluorescence measurement, each 20 μL. For the 4MU standard, a diluted standard solution diluted 2-fold from 2.5 μM was transferred to a 20 μL fluorescence measurement 96-well black microplate in the same manner as the blood sample. 40 μL of each substrate was added to a well containing the specimen and 4MU standard solution, and allowed to stand for 4 hours in each constant temperature bath at 25 ° C., 37 ° C., and 45 ° C. to perform an enzyme reaction. After completion of the enzyme reaction, add 200 μL of the common reaction stop solution to each well, mix gently, and then measure the fluorescence intensity at an excitation wavelength of 370 nm and a detection wavelength of 465 nm using a fluorescence autoreader (TECASN, infinite M200pro). The enzyme activity per dry blood filter paper was determined by comparison with the fluorescence intensity of the 4MU standard solution.
4MU標準品の蛍光強度の測定結果を図３に示した。また、この検量線より計算される酵素活性の結果と酵素反応の温度の関係を図４、図５及び図６に示した。GLAとGBAの酵素活性は、37℃付近に最適反応温度が示される一方で、GAAは37℃より45℃と、より高温で反応を行わせた方が酵素活性が高くなるという知見は驚くべきものであった。しかしながら、GAA酵素活性は、37℃で反応させた場合であっても、ポンペ病患者血液を模したcontrol（L）と比較して十分に高い値を示すため、反応温度の共通化は可能であることが示された。 The measurement results of the fluorescence intensity of the 4MU standard product are shown in FIG. Moreover, the relationship between the enzyme activity result calculated from the calibration curve and the temperature of the enzyme reaction is shown in FIG. 4, FIG. 5 and FIG. The enzyme activity of GLA and GBA shows an optimum reaction temperature around 37 ° C, while GAA is surprisingly high in enzyme activity when the reaction is carried out at a higher temperature of 45 ° C than 37 ° C. It was a thing. However, the GAA enzyme activity shows a sufficiently high value compared to control (L) simulating Pompe disease patient blood even when reacted at 37 ° C, so it is possible to make the reaction temperature common. It was shown that there is.
乾燥血液ろ紙を検体として、GLA、GAA、及びGBAの酵素活性を、酵素反応時間を変えて検出した。その方法は、酵素反応温度を37℃とした以外は、実施例１に示した方法と同様である。 Using dry blood filter paper as a specimen, the enzyme activities of GLA, GAA, and GBA were detected by changing the enzyme reaction time. The method is the same as the method shown in Example 1 except that the enzyme reaction temperature is 37 ° C.
酵素反応時間と酵素活性の関係を図７、図８及び図９に示した。GLAの酵素活性は、反応時間の経過と共に低下し、2時間で酵素反応を終了させた場合が最も高い活性を示した。一方、GAAとGBAの酵素活性は、2時間〜24時間の反応でほとんど変化はなかった。この結果より、本発明法では、従来の24時間の酵素反応を行わなくても、2時間〜4時間の反応で測定可能であることが示された。 The relationship between the enzyme reaction time and the enzyme activity is shown in FIG. 7, FIG. 8 and FIG. The enzyme activity of GLA decreased with the lapse of the reaction time, and the highest activity was shown when the enzyme reaction was completed in 2 hours. On the other hand, the enzyme activities of GAA and GBA remained almost unchanged during the 2-24 hour reaction. From these results, it was shown that the method of the present invention can be measured in a reaction of 2 hours to 4 hours without performing the conventional enzyme reaction for 24 hours.
乾燥血液ろ紙を検体としてGLA、GAA、及びGBAの酵素活性を、ファブリー病患者、ゴーシェ病患者、及びポンペ病患者と正常人とで比較した。その方法は、酵素反応温度を37℃、酵素反応時間を4時間とした以外は、実施例１に示した方法と同様である。 Using dry blood filter paper as a specimen, the enzyme activities of GLA, GAA, and GBA were compared among Fabry disease patients, Gaucher disease patients, Pompe disease patients, and normal people. The method is the same as the method shown in Example 1 except that the enzyme reaction temperature is 37 ° C. and the enzyme reaction time is 4 hours.
本実施例で使用した検体は、正常人（新生児乾燥血液ろ紙）10,226例、ファブリー病患者2例、ポンペ病患者1例、ゴーシェ病患者1例である。 The specimens used in this example were 10,226 normal persons (newborn dry blood filter paper), 2 Fabry disease patients, 1 Pompe disease patient, and 1 Gaucher disease patient.
正常人の各酵素活性の分布と各疾患患者の酵素活性を図１０、図１１及び図１２に示した。ファブリー病の責任酵素であるGLA酵素活性の正常新生児の平均酵素活性は、31.0pmol/hr/disk、ポンペ病の責任酵素であるGAA酵素活性の正常新生児の平均酵素活性は、29.9pmol/hr/disk、ゴーシェ病の責任酵素活性であるGBA酵素活性の正常新生児の平均酵素活性は16.1pmol/hr/diskであり、各疾患患者の酵素活性は、明らかに正常人の酵素活性分布より低い結果となった。従って、本発明の測定により、１枚の乾燥血液ろ紙を用いて、３種類の酵素の活性を同時に測定することにより、迅速な３疾患のマススクリーニング検査が可能となることが示された。 The distribution of each enzyme activity in normal persons and the enzyme activity in each disease patient are shown in FIG. 10, FIG. 11 and FIG. The average enzyme activity of normal neonates with GLA enzyme activity responsible for Fabry disease is 31.0 pmol / hr / disk, and the average enzyme activity of normal neonates with GAA enzyme activity responsible for Pompe disease is 29.9 pmol / hr / disk The average enzyme activity of normal neonates with GBA enzyme activity, which is the responsible enzyme activity of disk and Gaucher disease, is 16.1 pmol / hr / disk, and the enzyme activity of each disease patient is clearly lower than the distribution of enzyme activity of normal people became. Therefore, the measurement of the present invention showed that a rapid mass screening test for three diseases can be performed by simultaneously measuring the activity of three kinds of enzymes using one sheet of dry blood filter paper.
本発明による測定方法は、新生児マススクリーニング検査等において利用可能である。 The measurement method according to the present invention can be used in a newborn mass screening test or the like.
Claims (15)
（１）乾燥血液ろ紙から共通抽出液（25ｍMのリン酸イオン及び25ｍMのクエン酸イオンを含むpH5.5〜6.5の緩衝液で、0.1ｍM〜5ｍMのDithiothreitol（DTT）、マグネシウムイオン（Mg2+）、0.05％〜0.5％のTritonX-100、及び0.02％〜0.1％のアジ化ナトリウムを含む）を用いて血液試料を抽出する工程、
（２）抽出した血液試料に、4-Methylumbelliferyl合成基質を含む基質液（クエン酸-リン酸緩衝液）を添加して酵素反応を行わせる工程、
（３）酵素反応により得られた生成物を、励起波長365〜375nm、検出波長460〜470nmの測定波長で蛍光測定する工程。 A rapid mass screening method for lysosomal disease responsible enzyme that rapidly measures the activity of a plurality of lysosomal disease responsible enzymes from a single dry blood filter paper, wherein the responsible enzyme is α-D-galactosidase A (GLA), acidic An enzyme selected from the group consisting of α-glucosidase (GAA) and acidic β-glucosidase (GBA), comprising the following steps:
(1) Dry common extract from the blood filter paper (with a buffer pH5.5~6.5 containing phosphate ion and 25mM of citrate ion of 25mM, Dithiothreitol of 0.1 mm to 5 mm (DTT), magnesium ions (Mg 2+ ), 0.05% to 0.5% TritonX-100, and 0.02% to 0.1% sodium azide)
(2) a step of adding a substrate solution (citrate-phosphate buffer) containing 4-Methylumbelliferyl synthetic substrate to the extracted blood sample to perform an enzyme reaction;
(3) A step of measuring fluorescence of a product obtained by the enzyme reaction at an excitation wavelength of 365 to 375 nm and a detection wavelength of 460 to 470 nm.
（１）乾燥血液ろ紙、
（２）共通抽出液（0.1％TrtionX-100、5ｍM MgCl2、0.5ｍM DTT及び0.05％アジ化ナトリウムを含むpH6.0の25ｍMクエン酸-25ｍMリン酸カリウム緩衝液）、
（３）4-Methylumbelliferyl合成基質を含む基質液、
（４）共通反応停止液（300ｍMグリシン‐NaOH緩衝液（pH10.6））、及び
（５）酵素反応により得られた生成物を蛍光測定する手段。 A kit for use in the inspection method according to claim 1, comprising:
(1) dry blood filter paper,
(2) Common extract (0.1 mM TrtionX-100, 5 mM MgCl 2 , 0.5 mM DTT and 0.05% sodium azide, pH 6.0, 25 mM citrate-25 mM potassium phosphate buffer),
(3) a substrate solution containing a 4-Methylumbelliferyl synthetic substrate,
(4) Common reaction stop solution (300 mM glycine-NaOH buffer (pH 10.6)), and (5) Means for fluorescence measurement of the product obtained by the enzyme reaction.
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